Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are two fundamental cell lineages for cardiac repair via cardiomyogenesis and angiogenesis, respectively. While functional CMs are possible to be generated from pluripotent stem cells (PSCs) including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). MSCs derived from PSCs possess much higher self-renewal potential with superior functions in vivo. CMs and MSCs derived from patient-derived human induced pluripotent stem cells (hiPSCs) are immunocompatible and thus hold, a great potential in clinical applications. At present, CMs and MSCs are separately derived from hiPSCs or hESCs and the methods require cumbersome multi-step and expensive procedures that are poorly controlled. Moreover, the quality of MSCs could be compromised if the differentiation is initiated from poor hESCs/hiPSCs with spontaneous differentiation.
To date, different approaches have been adopted in generating MSCs from hESCs. In most of the cases, hESCs cultured with feeder-free conditions were directly differentiated into MSC-like cells in medium similar to that for hESCs but with the supplementation of more bovine fibroblast growth factor (bFGF) and epithelial growth factor (EGF). Homogeneous MSCs were obtained after several passaging and FACS-based cell sorting, a process that could take a few weeks.
Alternatively, hESC-MSCs were derived from Day 4 and 10 embryoid bodies (EBs) that were plated onto gelatin-coated tissue culture plates and the MSC-like cells migrated from EBs and were subcultured.
The homogeneity of hESC/hiPSC-derived MSCs is a key issue for the clinical application of hiPSC-MSCs as they are derived from highly pluripotent stem cells which are capable of teratoma formation in vivo. To address such concern, a more defined method for the generating of potentially clinical applicable MSCs was developed. In brief, hESCs and hiPSCs were directly differentiated in a defined medium containing growth factors, fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) or epidermal growth factor (EGF). Next, homogeneous MSCs were obtained either by FACS-based cell sorting for CD24−/CD105+ or by limited dilution to generate single-cell derived colonies. After all, all of the above methods were developed from established hESC lines and little details were given on the differentiation efficiency. In addition, these methods require expensive consumables (growth factors and antibodies) and high-end equipment (FACS sorter).
Since hiPSC colonies are generally considered as less stable than that of hESCs and are more prone to spontaneous differentiation, the generation of high quality hiPSC-MSCs could be more challenging. To date, only the method of Lian et al., 2007 (direct differentiation plus CD24−/CD105+ sorting) has been used to differentiate hiPSCs into MSCs.
Cardiac cell therapy requires not only functional cardiomyocytes (CMs), but also pro-angiogenic cell lineages such as mesenchymal stem/stromal cells (MSCs) for effective cardiac repair. However, clinical application of CMs has been hampered by the lack of autologous functional human CMs where cardiac biopsy failed to yield CMs that can be expanded in culture. On the other hand, MSCs derived from adult tissues (bone marrow and adipose tissue) have shown therapeutic potential. Currently, hiPSC-CM and hiPSC-MSCs are separately derived from disparate mesodermal differentiation protocols which are tedious and time-consuming. However, adult tissue-derived MSCs are limited in their clinical application due to their poor self-renewal potential and functional competency in the elderly individuals and in patients with chronic diseases. Although human embryonic stem cells (hESCs) are capable of generating functional CMs and MSCs, their clinical applications have been hampered by immunoreactivity and ethical issues.
An alternative means of deriving MSCs is desirable for successful therapeutic applications together with CMs, for example in autologous cardiac cell therapy.